title: The small GTPase RhoA localizes to the nucleus and is activated by Net1 and DNA damage signals.
abstract: BACKGROUND
  Rho GTPases control many cellular processes, including cell survival, gene expression and migration. Rho proteins reside mainly in the cytosol and are targeted to the plasma membrane (PM) upon specific activation by guanine nucleotide exchange factors (GEFs). Accordingly, most GEFs are also cytosolic or associated with the PM. However, Net1, a RhoA-specific GEF predominantly localizes to the cell nucleus at steady-state. Nuclear localization for Net1 has been seen as a mechanism for sequestering the GEF away from RhoA, effectively rendering the protein inactive. However, considering the prominence of nuclear Net1 and the fact that a biological stimulus that promotes Net1 translocation out the nucleus to the cytosol has yet to be discovered, we hypothesized that Net1 might have a previously unidentified function in the nucleus of cells.
  
  
  RESULTS
  Using an affinity precipitation method to pulldown the active form of Rho GEFs from different cellular fractions, we show here that nuclear Net1 does in fact exist in an active form, contrary to previous expectations. We further demonstrate that a fraction of RhoA resides in the nucleus, and can also be found in a GTP-bound active form and that Net1 plays a role in the activation of nuclear RhoA. In addition, we show that ionizing radiation (IR) specifically promotes the activation of the nuclear pool of RhoA in a Net1-dependent manner, while the cytoplasmic activity remains unchanged. Surprisingly, irradiating isolated nuclei alone also increases nuclear RhoA activity via Net1, suggesting that all the signals required for IR-induced nuclear RhoA signaling are contained within the nucleus.
  
  
  CONCLUSIONS
  These results demonstrate the existence of a functional Net1/RhoA signaling pathway within the nucleus of the cell and implicate them in the DNA damage response.
authors.affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
authors.forename: Adi D
authors.initials: AD
authors.lastname: Dubash
authors.forename: Christophe
authors.initials: C
authors.lastname: Guilluy
authors.forename: Melissa C
authors.initials: MC
authors.lastname: Srougi
authors.forename: Etienne
authors.initials: E
authors.lastname: Boulter
authors.forename: Keith
authors.initials: K
authors.lastname: Burridge
authors.forename: Rafael
authors.initials: R
authors.lastname: García-Mata
mesh_terms: D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004249:DNA Damage / Q000502:physiology*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014018:Tissue Distribution / Q000187:drug effects; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; C099763:NET1 protein, human; D015513:Oncogene Proteins; D034741:RNA, Small Interfering; D020559:Monomeric GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein